Protective role of steroids on mouse primary RPE cells under hypoxic stress

Abstract

AbstractPurpose Angiogenesis is a clinically critical aspect of the pathogenesis of many retinal neovascular diseases. Triamcinolone acetonide (TA) is an important anti‐angiogenic and anti‐inflammatory agent. This study has evaluated the effect of TA on the expression of pigment epithelium derived factor (PEDF), thrombospondin‐1 (TSP‐1) and vascular endothelial growth factor A (VEGF‐A) in cultured mouse RPE cells under hypoxic stress.Methods Primary cultures of mouse RPE cells were grown in culture to passage 2‐3 and were then subjected to hypoxic stress. TA (50µg/ml) was used to determine the steroid effect under normoxic (95% air/5 % carbon dioxide) and hypoxic (95% nitrogen/5% carbon dioxide) conditions. Hypoxia was continued for 48 hrs with and without TA. Cells were harvested at 0, 24 and 48 hours and both RNA and protein were extracted. Real Time PCR was used to analyze expression levels of PEDF, TSP‐1 and VEGF‐A. The expression pattern of PEDF, TSP‐1 and VEGF‐A in the presence of TA were quantifie.Results After 24 hours of hypoxia, mRNA expression levels of both PEDF and TSP‐1 were down‐regulated by 2‐fold. Hypoxic stress for 48 hours, resulted in 2.2‐fold down‐regulation of PEDF and a stronger down‐regulation of TSP‐1 of 3.4‐fold. VEGF‐A transcripts levels, on the other hand, showed an opposite response to hypoxic stress, an up‐regulation of 1.9‐fold at 24 hours and 2.7‐fold increase at 48 hours. Hypoxic RPE cells, when exposed to TA, led to the rapid recovery of PEDF and TSP‐1 transcripts at 24 hours after the initiation of hypoxia. VEGF‐A mRNA expression rebounded to control levels in TA treated hypoxic cells. Western blots showed that TSP‐1 protein levels correlated with the PCR results. TA significantly induced the expression of TSP‐1 protein in hypoxic cells, and the strongest expression was at 48 hours.Conclusion The results suggests that TA has a protective effect on PEDF and TSP‐1 levels, and on the fine balance of PEDF, TSP‐1 and VEGF‐A levels.